Because an amino acid can be encoded by as many as six codons, there are many different ways to encode any particular protein. Biases exist in the way codons are used. A little-studied bias is the codon pair bias, such that some codons prefer to be adjacent to certain other codons. This codon pair bias is completely separate and independent from codon usage. Recently, we have found that when viruses are re-coded to have a bad codon pair bias, the viruses are attenuated, in extreme cases to inviability. It appears that attenuation of a virus via a bad codon pair bias can be used to make a live, attenuated vaccine. However, while the procedure works, nothing whatever is known about the mechanism by which codon pair bias causes attenuation. In this proposal, we will investigate the mechanisms of attenuation by bad codon pair bias. This will be done in yeast, where we have recently shown there are strong codon pair bias effects. We have found two major classes of attenuating codon pairs, and each class may work by a distinct mechanism. Recent results suggest that the attenuating codon pairs are interfering with proper translation, which is triggering translation quality control pathways such as nonsense mediated decay to destroy the recoded mRNAs. Here, we will learn the mechanisms of attenuation by codon pair bias. The significance of this is twofold: from a basic research point-of-view, this research may reveal completely new aspects of RNA processing and translational quality control explaining how a change in the ordering of synonymous codons can drastically affect gene expression; from a medical point-of-view, this research will help us design and synthesize better attenuated viruses for potential use in vaccines. Relevance One way to make an anti-viral vaccine is to mutate the virus to weaken it, and then use this weakened virus as a vaccine (e.g., FluMist, a live Flu vaccine). However there are many difficulties. Recently we have found a new method, codon pair de-optimization, for making live attenuated viral vaccines. Although the method works (for polio, flu, dengue, respiratory syncytial virus), we do not know why it works. Here, we will study the mechanism of attenuation by codon pair de-optimization.